Identification of a Zn2+ binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of beta subunits.
1. Whole-cell currents were recorded from Xenopus laevis oocytes expressing wild-type and mutant recombinant GABAA receptors to locate a binding site for Zn2+ ions in the beta 3 subunit. 2. The Cl(-)-selective current, spontaneously gated by beta 3 subunit homomers, was enhanced by pentobarbitone and inhibited by picrotoxinin. The potencies of these agents were minimally affected by mutating histidine (H) 292 to alanine (A) in the second transmembrane domain (TM2). 3. Zn2+ inhibited the beta 3 subunit-gated conductance (IC50, 0.31 microM); the inhibition was voltage insensitive. The H292A mutation in beta 3 subunits caused a 1000-fold reduction in Zn2+ potency (IC50, 307 microM). 4. GABA-activated responses recorded from heteromeric alpha 1 beta 3 GABAA receptors were also inhibited by Zn2+ (IC50, 0.11 microM). This inhibition was reduced by mutating H292A in the beta 3 subunit (IC50, 22.8 microM). 5. H292 in TM2 of the beta 3 subunit is an important determinant of a Zn2+ binding site on the GABAA receptor. Its location in the presumed ion channel lining suggests that Zn2+ can penetrate into an anion-selective channel and that the ionic selectivity filter and channel gate are located beyond H292.
Wooltorton
J R
JR
Department of Pharmacology, School of Pharmacy, London, UK.
McDonald
B J
BJ
Moss
S J
SJ
Smart
T G
TG
D019943:Amino Acid Substitution; D000818:Animals; D006639:Histidine / Q000235:genetics; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D018408:Patch-Clamp Techniques; D051381:Rats; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D007473:Ion Channels; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D006639:Histidine; D015032:Zinc